JP2023094881A - Method for producing culture supernatant - Google Patents
Method for producing culture supernatant Download PDFInfo
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- JP2023094881A JP2023094881A JP2021210450A JP2021210450A JP2023094881A JP 2023094881 A JP2023094881 A JP 2023094881A JP 2021210450 A JP2021210450 A JP 2021210450A JP 2021210450 A JP2021210450 A JP 2021210450A JP 2023094881 A JP2023094881 A JP 2023094881A
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Abstract
Description
本発明は、培養上清を生産する方法、並びに培養上清においてサイトカイン及び/又はエクソソームの含有量を増加させる方法に関する。 The present invention relates to methods of producing culture supernatants and methods of increasing the content of cytokines and/or exosomes in culture supernatants.
間葉系幹細胞の培養上清には、サイトカインやエクソソーム等の生物活性物質が含まれており、損傷治癒や免疫抑制効果等の治療効果を有することが知られている。培養上清の治療効果を高めるために、培養上清中の生物活性物質含量を増大させる技術が重要である。 The culture supernatant of mesenchymal stem cells contains bioactive substances such as cytokines and exosomes, and is known to have therapeutic effects such as wound healing and immunosuppressive effects. Techniques for increasing the content of biologically active substances in culture supernatants are important in order to enhance the therapeutic efficacy of culture supernatants.
培養上清において生物活性物質の含有量を向上させる方法としては、細胞を薬物やホルモン等で刺激する方法が最も簡便であると考えられる。しかしながら、この方法では薬物やホルモン等が培養上清中に残留し得るため、回収した培養上清を生体に直接適用することはできない。それ故、有害成分を除去する追加の工程が必要となってしまうため、この方法は必ずしも簡便とは言い難い。 As a method for increasing the content of biologically active substances in the culture supernatant, stimulating cells with drugs, hormones, etc. is considered to be the simplest method. However, since drugs, hormones, and the like may remain in the culture supernatant by this method, the collected culture supernatant cannot be directly applied to the living body. Therefore, this method is not always convenient because it requires an additional step to remove harmful components.
特許文献1は、ウシ胎児血清等の血清成分を添加した培地で継代培養を行った後、非ヒトタンパク質不含培地で細胞を培養する前培養工程を行うことによって、培養上清を生産する本培養への血清成分の混入を防ぐ方法を開示している。特許文献1には、培養上清中の生物活性物質含有量をさらに向上させるための方法や、薬物やホルモン等に依らずこれを達成する方法は開示されていない。 In Patent Document 1, culture supernatant is produced by performing subculture in a medium supplemented with serum components such as fetal bovine serum and then performing a pre-culture step of culturing cells in a non-human protein-free medium. A method for preventing contamination of the main culture with serum components is disclosed. Patent document 1 does not disclose a method for further increasing the content of biologically active substances in the culture supernatant, nor a method for achieving this without relying on drugs, hormones, or the like.
したがって、培養上清中の生物活性物質含有量を簡便に向上させ得る技術が必要とされている。 Therefore, there is a need for a technique that can easily improve the content of biologically active substances in culture supernatants.
本発明の目的は、培養上清中の生物活性物質含有量を簡便に向上させる新たな方法を提供することである。 An object of the present invention is to provide a new method for simply increasing the content of biologically active substances in culture supernatants.
生物活性物質を含む培養上清を生産する従来の方法では、生育に適した条件下で継代培養された、増殖状態のよい細胞を用いることが一般的に有利であると考えられてきた。それ故、保存していた凍結細胞を解凍して培養上清を生産する場合、少なくとも数代の継代を行った後に培養上清を生産することが技術常識となっていた。 In previous methods of producing culture supernatants containing biologically active substances, it has generally been considered advantageous to use well-growing cells that have been subcultured under conditions suitable for growth. Therefore, when producing a culture supernatant by thawing stored frozen cells, it has become common general knowledge to produce the culture supernatant after at least several passages.
本発明者らは、上記課題を解決するため、凍結した細胞を解凍して培養上清を生産する際に、生物活性物質含有量が向上し得る条件について検討した。その結果、従来の技術常識に反して解凍後の継代数が少ない細胞から、生物活性物質含有量が向上した培養上清が得られるという驚くべき効果を見出した。本発明の方法では薬物やホルモン等による刺激が必要ないため、培養上清の安全性が担保される点も極めて有利である。本発明は、上記研究成果に基づくものであって、以下を提供する。 In order to solve the above-mentioned problems, the present inventors examined conditions under which the biologically active substance content can be improved when frozen cells are thawed to produce a culture supernatant. As a result, the inventors have found a surprising effect that, contrary to conventional technical knowledge, a culture supernatant with an improved content of biologically active substances can be obtained from cells with a small passage number after thawing. Since the method of the present invention does not require stimulation with drugs, hormones, etc., it is extremely advantageous in that the safety of the culture supernatant is ensured. The present invention is based on the above research results and provides the following.
(1)培養上清を生産する方法であって、
凍結した細胞を解凍する、解凍工程、
前記細胞を第一の培地で培養する、第一培養工程、
前記第一培養工程後に得られる培養上清を除去する、除去工程、
前記除去工程後の前記細胞をさらに第二の培地で培養する、第二培養工程、及び
前記第二培養工程後に得られる培養上清を回収する、回収工程
を含み、
前記第一の培地が、基本培地又は無血清培地であり、かつ
前記第二の培地が、タンパク質を含まない、前記方法。
(2)前記第一の培地が、ヒトタンパク質又は非ヒトタンパク質を含む、(1)に記載の方法。
(3)前記基本培地が、EMEM培地、DMEM培地、IMDM培地、GMEM培地、ハムF10培地、ハムF12培地、RPMI1640培地、及びこれらの組み合わせからなる群から選択される、(1)又は(2)に記載の方法。
(4)前記無血清培地が、MesenCult、StemPro MSC、BMN211、StemMACS、MSC NutriStem XF、Xuri NSC、PRIME-XV、StemXVivo、Human Mesenchymal-XF Expansion Medium、stemgro、STK2、PLTMax、ProculAD、Mesenchymal Stem Cell Growth Medium XF、Mesenchymal Stem Cell Growth Medium 2、MesenGro、StemFit、CiMS-BM、MSC-Brew GMP Medium、StemXVivo Serum-Free Human MSC Expansion Media、MSC-T4、及びこれらの任意の組み合わせからなる群から選択される、(1)又は(2)に記載の方法。
(5)前記第一培養工程を12時間~120時間行う、(1)~(4)のいずれかに記載の方法。
(6)前記解凍工程後、前記第一培養工程の前に、血清又は血漿含有培地で前記細胞を培養して増殖させる増殖工程をさらに含む、(1)~(5)のいずれかに記載の方法。
(7)前記増殖工程を1回以上繰り返す繰り返し工程を含む、(6)に記載の方法。
(8)前記除去工程後、前記第二培養工程前に前記細胞を洗浄する洗浄工程をさらに含む、(1)~(7)のいずれかに記載の方法。
(9)前記第二の培地が基本培地である、(1)~(8)のいずれかに記載の方法。
(10)前記解凍工程の前に、
前培養培地で細胞を培養する、前培養工程、
前記前培養工程後の前記前培養培地を凍結保存液と置換する、置換工程、及び
前記置換工程後の前記細胞を凍結する、凍結工程
を含み、
前記前培養培地が、血清又は血漿含有培地、無血清培地、又は基本培地である、(1)~(9)のいずれかに記載の方法。
(11)前記前培養培地が、ヒトタンパク質又は非ヒトタンパク質を含む、(10)に記載の方法。
(12)前記血清が、動物由来であるか、又は人工血清である、(10)又は(11)に記載の方法。
(13)前記動物由来の血清が、ウシ胎児血清、ウシ新生児血清、ウシ血清、及びウマ血清からなる群から選択される、(12)に記載の方法。
(14)前記細胞が間葉系幹細胞である、(1)~(13)のいずれかに記載の方法。
(15)前記第一培養工程を12時間~48時間行う、(14)に記載の方法。
(16)(1)~(15)のいずれかに記載の方法により生産される培養上清を含む、組成物。
(17)培養上清においてサイトカイン及び/又はエクソソームの含有量を増加させる方法であって、
凍結した細胞を解凍する、解凍工程、
前記細胞を第一の培地で培養する、第一培養工程、
前記第一培養工程後に得られる培養上清を除去する、除去工程、
前記除去工程後の前記細胞をさらに第二の培地で培養する、第二培養工程、及び
前記第二培養工程後に得られるサイトカイン及び/又はエクソソームを含む培養上清を回収する、回収工程
を含み、
前記第一の培地が、基本培地又は無血清培地であり、かつ
前記第二の培地が、タンパク質を含まない、前記方法。
(1) A method for producing a culture supernatant,
thawing the frozen cells, a thawing step;
a first culturing step of culturing the cells in a first medium;
a removing step of removing the culture supernatant obtained after the first culturing step;
a second culturing step of further culturing the cells after the removing step in a second medium; and a collecting step of collecting the culture supernatant obtained after the second culturing step,
The method as described above, wherein the first medium is a basal medium or a serum-free medium, and the second medium is protein-free.
(2) The method of (1), wherein the first medium contains a human protein or a non-human protein.
(3) the basal medium is selected from the group consisting of EMEM medium, DMEM medium, IMDM medium, GMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, and combinations thereof, (1) or (2); The method described in .
(4) the serum-free medium is MesenCult, StemPro MSC, BMN211, StemMACS, MSC NutriStem XF, Xuri NSC, PRIME-XV, StemXVivo, Human Mesenchymal-XF Expansion Medium, stemgro, STK2, PLTMax, ProculAD, Mesenchymal Stem Cell Growth selected from the group consisting of Medium XF, Mesenchymal Stem Cell Growth Medium 2, MesenGro, StemFit, CiMS-BM, MSC-Brew GMP Medium, StemXVivo Serum-Free Human MSC Expansion Media, MSC-T4, and any combination thereof , (1) or (2).
(5) The method according to any one of (1) to (4), wherein the first culture step is performed for 12 hours to 120 hours.
(6) The method according to any one of (1) to (5), further comprising, after the thawing step and before the first culturing step, a growing step of culturing and growing the cells in a serum- or plasma-containing medium. Method.
(7) The method according to (6), comprising repeating the growing step one or more times.
(8) The method according to any one of (1) to (7), further comprising a washing step of washing the cells after the removing step and before the second culturing step.
(9) The method according to any one of (1) to (8), wherein the second medium is a basal medium.
(10) before the thawing step,
a pre-culture step of culturing cells in a pre-culture medium;
A replacement step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution, and a freezing step of freezing the cells after the replacement step,
The method according to any one of (1) to (9), wherein the pre-culture medium is serum- or plasma-containing medium, serum-free medium, or basal medium.
(11) The method of (10), wherein the pre-culture medium contains human or non-human proteins.
(12) The method according to (10) or (11), wherein the serum is animal-derived or artificial serum.
(13) The method according to (12), wherein the animal-derived serum is selected from the group consisting of fetal bovine serum, newborn bovine serum, bovine serum, and horse serum.
(14) The method according to any one of (1) to (13), wherein the cells are mesenchymal stem cells.
(15) The method according to (14), wherein the first culturing step is carried out for 12 to 48 hours.
(16) A composition comprising a culture supernatant produced by the method according to any one of (1) to (15).
(17) A method for increasing the content of cytokines and / or exosomes in the culture supernatant,
thawing the frozen cells, a thawing step;
a first culturing step of culturing the cells in a first medium;
a removing step of removing the culture supernatant obtained after the first culturing step;
Further culturing the cells after the removal step in a second medium, a second culturing step, and recovering the culture supernatant containing cytokines and / or exosomes obtained after the second culturing step, including a recovery step,
The method as described above, wherein the first medium is a basal medium or a serum-free medium, and the second medium is protein-free.
本発明によれば、培養上清中の生物活性物質含有量を簡便に向上させる新たな方法が提供される。 INDUSTRIAL APPLICABILITY According to the present invention, a new method is provided for simply increasing the content of biologically active substances in culture supernatants.
<培養上清を生産する方法>
一態様において、本発明は培養上清を生産する方法に関する。本発明の培養上清の生産方法は、解凍工程、第一培養工程、除去工程、第二培養工程、及び回収工程を必須工程として含む。また、選択工程として、増殖工程、繰り返し工程、洗浄工程、及び精製工程等の他の工程を含むことができる。本態様の方法の一実施形態は図1に図示される。本態様の方法は、さらなる選択工程として、解凍工程の前に、前培養工程、置換工程、及び凍結工程を含む(図2)。本発明の方法を構成する各工程について、以下詳細に説明する。
<Method for producing culture supernatant>
In one aspect, the invention relates to a method of producing a culture supernatant. The method for producing a culture supernatant of the present invention includes, as essential steps, a thawing step, a first culturing step, a removal step, a second culturing step, and a recovery step. Selection steps can also include other steps such as growth steps, cycling steps, washing steps, and purification steps. One embodiment of the method of this aspect is illustrated in FIG. The method of this embodiment includes, as additional selection steps, a pre-incubation step, a replacement step, and a freezing step prior to the thawing step (Figure 2). Each step constituting the method of the present invention is described in detail below.
(前培養工程)
本態様の方法は、前培養培地で細胞を培養する前培養工程を選択工程として含む。本明細書において「前培養培地」とは、前培養工程で細胞を培養するために用いる培地をいう。前培養培地は、血清若しくは血漿含有培地、基本培地、又は無血清培地である。
(Pre-culture step)
The method of this embodiment includes a pre-culture step of culturing cells in a pre-culture medium as a selection step. As used herein, the term "pre-culture medium" refers to a medium used for culturing cells in the pre-culture step. Pre-culture media are serum- or plasma-containing media, basal media, or serum-free media.
本態様の方法において、血清又は血漿含有培地に含まれる血清又は血漿は、動物由来であるか、又は人工血清であってもよい。動物は後述する動物の例のいずれであってもよく、例えばヒトやヒト以外の動物(例えば、ウシ又はウマ等の哺乳動物)が挙げられる。ヒト由来の血清又は血漿として、ヒト血清、ヒト血漿、多血小板血漿(PRP、Platelet rich plasma)、及びヒトAB血清(ヒトAB型由来の血清)が例示される。また、ヒト以外の動物に由来する血清又は血漿の例として、ウシ胎児血清、ウシ新生児血清、ウシ血清、及びウマ血清が挙げられる。 In the method of this aspect, the serum or plasma contained in the serum- or plasma-containing medium may be animal-derived or artificial serum. The animal may be any of the examples of animals described below, including humans and non-human animals (eg, mammals such as cows and horses). Examples of human-derived serum or plasma include human serum, human plasma, platelet rich plasma (PRP), and human AB serum (serum derived from human type AB). Examples of serum or plasma derived from non-human animals also include fetal bovine serum, newborn bovine serum, bovine serum, and equine serum.
本明細書において「人工血清」とは、血清を代替し得るが、動物由来又は異種動物由来の成分を含まない培地添加剤をいう。人工血清は、培地に添加して細胞を培養する場合に、動物由来の血清と同等又はそれ以上の増殖性能を示し得る。人工血清の具体例として、Artificial Serum (Xeno-free)(細胞科学研究所、#87-081)、Artificial Serum (Animal-free)(細胞科学研究所、#87-082)、及びStemSure血清代替品(富士フイルム和光純薬(株)、#191-18375)が挙げられる。 As used herein, the term "artificial serum" refers to a medium additive that can substitute for serum but does not contain animal-derived or heterologous animal-derived components. When artificial serum is added to a medium and cells are cultured, it can exhibit growth performance equivalent to or superior to animal-derived serum. Specific examples of artificial sera include Artificial Serum (Xeno-free) (Cell Science Research Institute, #87-081), Artificial Serum (Animal-free) (Cell Science Research Institute, #87-082), and StemSure serum substitutes. (Fuji Film Wako Pure Chemical Industries, Ltd., #191-18375).
本明細書において、「基本培地」とは、細胞が生存するために必要な成分、例えば無機塩類、必須アミノ酸、及びビタミン類、並びに緩衝剤等を含んだ溶液を意味する。基本培地の例として、限定するものではないが、EMEM培地(αMEM培地とも記載される)、DMEM培地、IMDM培地、GMEM培地、ハムF10培地、ハムF12培地、RPMI1640培地、及びこれらの組み合わせが挙げられる。 As used herein, the term "basal medium" means a solution containing components necessary for cell survival, such as inorganic salts, essential amino acids, vitamins, buffers, and the like. Examples of basal media include, but are not limited to, EMEM medium (also described as αMEM medium), DMEM medium, IMDM medium, GMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI1640 medium, and combinations thereof. be done.
本明細書において、「無血清培地」とは、細胞の生存や増殖に必要な成分を含むが、無調整又は未精製の血清を含まない培地をいう。無血清培地に含まれ得る生存や増殖に必要な成分として、ホルモンや増殖因子の他、血清アルブミン、トランスフェリン、脂肪酸、コラーゲン前駆体、微量元素、2-メルカプトエタノール又は3’チオールグリセロール等が挙げられる。また、無血清培地は、精製された血液由来成分や動物組織由来成分であれば含むことができる。無血清培地の例として、限定するものではないが、MesenCult、StemPro MSC、BMN211、StemMACS、MSC NutriStem XF、Xuri NSC、PRIME-XV、StemXVivo、Human Mesenchymal-XF Expansion Medium、stemgro、STK2、PLTMax、ProculAD、Mesenchymal Stem Cell Growth Medium XF、Mesenchymal Stem Cell Growth Medium 2、MesenGro、StemFit、MSC-T4、CiMS-BM、MSC-Brew GMP Medium、StemXVivo Serum-Free Human MSC Expansion Media、及びこれらの任意の組み合わせが挙げられる。なお、上述の人工血清を含む培地は血清含有培地に包含されるものとする。 As used herein, the term "serum-free medium" refers to a medium that contains components necessary for cell survival and growth but does not contain unadjusted or unpurified serum. In addition to hormones and growth factors, serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3′ thiolglycerol, and the like can be mentioned as components necessary for survival and growth that can be contained in the serum-free medium. . In addition, the serum-free medium can contain purified blood-derived components and animal tissue-derived components. Examples of serum-free media include, but are not limited to, MesenCult, StemPro MSC, BMN211, StemMACS, MSC NutriStem XF, Xuri NSC, PRIME-XV, StemXVivo, Human Mesenchymal-XF Expansion Medium, stemgro, STK2, PLTMax, ProculAD , Mesenchymal Stem Cell Growth Medium XF, Mesenchymal Stem Cell Growth Medium 2, MesenGro, StemFit, MSC-T4, CiMS-BM, MSC-Brew GMP Medium, StemXVivo Serum-Free Human MSC Expansion Media, and any combination thereof be done. In addition, the medium containing the artificial serum described above is included in the serum-containing medium.
一実施形態において、前培養培地はタンパク質を含む。前培養培地がタンパク質を含むことにより、本工程において細胞の増殖、接着、及び分化誘導(又は未分化維持)等を促進することができる。タンパク質は、限定するものではないが、例えば細胞増殖因子、接着分子、及び分化誘導因子(又は分化抑制因子)であってもよい。タンパク質の具体例としては、線維芽細胞増殖因子(FGF)、血管内皮細胞増殖因子(VEGF)、肝細胞増殖因子(HGF)、上皮成長因子(EGF)、トランスフォーミング増殖因子-α(TGF-α)、トランスフォーミング増殖因子-β(TGF-β)、内皮細胞増殖因子(ECGF)、インスリン様増殖因子(IGF)、血小板由来増殖因子(PDGF)、骨形成因子(BMP)、フィブロネクチン、及びビトロネクチン等が挙げられる。 前培養培地に含まれ得るタンパク質は、ヒトに由来するタンパク質(本明細書において「ヒトタンパク質」という)、又は非ヒト生物に由来するタンパク質(本明細書において「非ヒトタンパク質」という)のいずれであってもよい。ヒトタンパク質は、ヒトから単離されてもよいし、ヒトに由来するタンパク質の組換え体を、哺乳動物、昆虫、酵母、及び大腸菌等の遺伝子組換え体に発現させて得てもよい。同様に、非ヒトタンパク質は、その非ヒトタンパク質が由来する生物種から単離されてもよいし、その生物種に由来するタンパク質の組換え体を、哺乳動物、昆虫、酵母、及び大腸菌等の遺伝子組換え体に発現させて得てもよい。非ヒトタンパク質の例としては、ラット、マウス、モルモット、ハムスター、ブタ、ウシ、ウマ、ヤギ、ヒツジ、ウサギ、イヌ、ネコ、及びサル等の哺乳動物、アフリカツメガエル等の両生類、ニワトリ及びダチョウ等の鳥類、昆虫類、線虫、酵母、大腸菌、及び植物からなる群より選択される生物、好ましくは非ヒト哺乳動物のタンパク質が挙げられる。 In one embodiment, the pre-culture medium comprises protein. By including protein in the pre-culture medium, it is possible to promote cell growth, adhesion, differentiation induction (or undifferentiated maintenance) and the like in this step. Proteins may be, but are not limited to, cell growth factors, adhesion molecules, and differentiation-inducing factors (or differentiation-inhibiting factors). Specific examples of proteins include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α ), transforming growth factor-β (TGF-β), endothelial cell growth factor (ECGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP), fibronectin, vitronectin, etc. is mentioned. The protein that can be contained in the pre-culture medium is either a human-derived protein (herein referred to as "human protein") or a non-human organism-derived protein (herein referred to as "non-human protein"). There may be. Human proteins may be isolated from humans, or recombinant proteins derived from humans may be expressed in genetic recombinants such as mammals, insects, yeast, and E. coli. Similarly, a non-human protein may be isolated from the species from which the non-human protein is derived, or a recombinant protein derived from that species may be isolated from mammals, insects, yeast, and E. coli. It may be obtained by expression in a gene recombinant. Examples of non-human proteins include mammals such as rats, mice, guinea pigs, hamsters, pigs, cows, horses, goats, sheep, rabbits, dogs, cats, and monkeys; amphibians such as Xenopus; Examples include proteins of organisms, preferably non-human mammals, selected from the group consisting of birds, insects, nematodes, yeast, E. coli, and plants.
別の実施形態において、前培養培地はタンパク質を含まない。
本発明の方法において、用いられる細胞の由来となる生物種は限定されず、例えばラット、マウス、モルモット、ハムスター、ブタ、ウシ、ウマ、ヤギ、ヒツジ、ウサギ、イヌ、ネコ、サル、及びヒト等の哺乳動物、好ましくはヒトである。また、用いられる具体的な細胞種も特定されず、例えば、ES細胞、iPS細胞、体性幹細胞、間葉系幹細胞(例えば歯髄由来幹細胞、脂肪由来幹細胞、臍帯由来幹細胞、臍帯血由来幹細胞、絨毛膜絨毛由来幹細胞、絨毛膜板由来幹細胞、脱落膜由来幹細胞、羊膜由来幹細胞、又は骨髄由来幹細胞)、線維芽細胞、皮膚細胞、肝細胞、膵細胞、神経細胞、軟骨細胞、内皮細胞、上皮細胞、骨細胞、筋細胞、生殖細胞、及び血液細胞等であってよい。細胞は初代培養細胞又は細胞株であってよく、また遺伝子組換えを行った細胞であっても、行っていない細胞であってもよい。これらの細胞の形状や構造は限定せず、例えば平面状に結合したシート形状や球状に多層化した凝集体であってもよい。
In another embodiment, the pre-culture medium does not contain protein.
In the method of the present invention, the biological species from which the cells used are derived is not limited, for example, rat, mouse, guinea pig, hamster, pig, cow, horse, goat, sheep, rabbit, dog, cat, monkey, human, and the like. mammals, preferably humans. In addition, the specific cell types used are not specified. villus-derived stem cells, chorionic plate-derived stem cells, decidua-derived stem cells, amnion-derived stem cells, or bone marrow-derived stem cells), fibroblasts, skin cells, hepatocytes, pancreatic cells, nerve cells, chondrocytes, endothelial cells, epithelial cells , bone cells, muscle cells, germ cells, blood cells, and the like. The cells may be primary cultured cells or cell lines, and may or may not be genetically modified cells. The shape and structure of these cells are not limited.
前培養工程の培養条件は限定されず、例えば、培養温度は約30℃~約40℃、CO2濃度は約2%~約10%であってよく、接着培養であっても懸濁培養であってもよい。 The culture conditions of the pre-culture step are not limited, for example, the culture temperature may be about 30° C. to about 40° C., the CO 2 concentration may be about 2% to about 10%, and even adhesion culture may be suspension culture. There may be.
また、前培養工程の時間は、特に限定しない。例えば、前培養工程は、数時間~数日行ってもよいし、拡大培養又は継代培養を経て数日~数週間又は数か月行ってもよい。なお、本明細書において、「継代」とは、細胞集団から一部を取り出して新しい培地に移し、細胞の増殖を維持する過程を意味する。 Moreover, the time for the pre-culture step is not particularly limited. For example, the pre-culturing step may be carried out for several hours to several days, or may be carried out for several days to several weeks or several months via expansion or subculture. As used herein, the term "passaging" means a process of removing a portion of a cell population, transferring it to a new medium, and maintaining cell growth.
(置換工程)
本態様の方法は、前培養工程後の前培養培地を凍結保存液と置換する置換工程を選択工程として含む。置換工程は、前培養培地を除去して凍結保存液を細胞に添加する工程である。前培養培地の除去は、デカンテーション及びアスピレーション等の通常の方法で行うことができる。
(Replacement step)
The method of this aspect includes a replacement step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution as a selection step. The replacement step is the step of removing the pre-culture medium and adding a cryopreservation medium to the cells. Removal of the pre-culture medium can be performed by conventional methods such as decantation and aspiration.
本明細書において、「凍結保存液」とは、細胞に対して凍結保護作用を示す任意の凍結保護剤を含む液体をいう。凍結保護剤の例として、限定するものではないが、ジメチルスルホキシド(DMSO)、グリセロール、エチレングリコール、プロピレングリコール、セリシン、プロパンジオール、デキストラン、ポリビニルピロリドン、ポリビニルアルコール、ヒドロキシエチルデンプン、コンドロイチン硫酸、ポリエチレングリコール、ホルムアミド、アセトアミド、カルボキシル化ポリリジン、アドニトール、ペルセイトール、ラフィノース、ラクトース、トレハロース、スクロース、及びマンニトール、並びにその任意の組み合わせが挙げられる。凍結保存液に含まれる凍結保護剤の濃度は、凍結保護剤の種類によって当業者が容易に決定できる。例えば0.1%~20%(v/v)、1%~15%(v/v)、又は2%~10%(v/v)であってもよい。 As used herein, the term “cryopreservation solution” refers to a liquid containing any cryoprotectant that exhibits a cryoprotective effect on cells. Examples of cryoprotectants include, but are not limited to, dimethylsulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol. , formamide, acetamide, carboxylated polylysine, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, and mannitol, and any combination thereof. The concentration of the cryoprotectant contained in the cryopreservation solution can be easily determined by those skilled in the art depending on the type of cryoprotectant. For example, 0.1% to 20% (v/v), 1% to 15% (v/v), or 2% to 10% (v/v).
また、前培養培地の除去と凍結保存液の添加の間に細胞を洗浄液で洗浄してもよい。洗浄液には、必要に応じて糖等の添加物を加えたバッファーや生理食塩水、及び/又は凍結保存液等を使用することができる。洗浄の回数は、例えば1回、2回、3回、又は4回とすることができる。 Alternatively, the cells may be washed with a washing solution between the removal of the pre-culture medium and the addition of the cryopreservation solution. As the washing solution, a buffer containing an additive such as sugar, a physiological saline solution, and/or a cryopreservation solution can be used, if necessary. The number of washes can be, for example, 1, 2, 3, or 4 times.
本工程において前培養培地を凍結保存液と置換することによって、後述の凍結工程及び解凍工程後の細胞の生存率や増殖能の低下を防ぐことができる。 By replacing the pre-culture medium with a cryopreservation medium in this step, it is possible to prevent deterioration of cell viability and proliferative ability after the below-described freezing and thawing steps.
(凍結工程)
本態様の方法は、置換工程後の細胞を凍結する凍結工程を選択工程として含む。
本工程において細胞を凍結する方法は限定せず、細胞を含む凍結保存液をクライオチューブ等の凍結保存用容器等に入れて、凍結保存液が凍結し得る温度以下に冷却すればよい。冷却手段は、限定しないが、例えば、ディープフリーザー等のフリーザーや、液体窒素等の低温の媒体を用いることができる。冷却温度は、凍結保存液の凍結温度以下であればよい。具体的な冷却温度は、緩慢凍結法やガラス化凍結法等の凍結方法によって適宜選択することができる。本明細書において「緩慢凍結法」とは、細胞内に凍結保護剤を浸透させた後、温度を徐々に低下させることにより細胞を凍結させる方法をいう。緩慢凍結法での冷却温度は、例えば、0℃以下、-10℃以下、-20℃以下、-30℃以下、-40℃以下、-50℃以下、-60℃以下、-70℃以下、又は-80℃以下であってもよい。本明細書において「ガラス化凍結法」とは、ガラス化を用いて細胞を凍結させる方法をいう。「ガラス化」とは、冷却速度や圧力等の冷却時の条件によって液体が結晶化せず固体となる現象であり、水も条件によってはガラス化することが知られている。ガラス化凍結法は、緩慢凍結法による効率的な保存が難しい場合に多く用いられている。ガラス化凍結法での冷却温度は、例えば-100℃以下、-110℃以下、-120℃以下、-130℃以下、-140℃以下、-150℃以下、-160℃以下、-170℃以下、-180℃以下、-190℃以下、又は-200℃以下である。冷却速度は、凍結解凍後の細胞生存率を大きく損なわない範囲であれば、限定せず、緩慢凍結法やガラス化凍結法等の凍結方法によって適宜選択することができる。緩慢凍結法であれば、例えば-1℃/分~-0.1℃/分、-0.8℃/分~-0.2℃/分、又は-0.6℃/分~-0.3℃/分、好ましくは-0.5℃/分~-0.4℃/分であってもよい。このような冷却速度は、クライオチューブ等の凍結保存用容器等をさらなる凍結処理容器に収容して、ディープフリーザーや液体窒素での冷却に供すれば達成することができる。ガラス化凍結法であれば、-5℃/分~-250℃/分、-10℃/分~-240℃/分、-15℃/分~-220℃/分、-20℃/分~-200℃/分、-50℃/分~-180℃/分、-100℃/分~-160℃/分、又は-120℃/分~-150℃/分であってもよく、液体窒素等を用いた急速冷凍が好ましく使用される。凍結工程後の細胞は、後述の解凍工程まで凍結状態で維持することができる。
(Freezing process)
The method of this embodiment includes a freezing step of freezing the cells after the replacement step as a selection step.
The method of freezing the cells in this step is not limited, and a cryopreservation solution containing cells may be placed in a cryopreservation container such as a cryotube and cooled to a temperature below which the cryopreservation solution can be frozen. Although the cooling means is not limited, for example, a freezer such as a deep freezer or a low temperature medium such as liquid nitrogen can be used. The cooling temperature may be lower than the freezing temperature of the cryopreservation solution. A specific cooling temperature can be appropriately selected according to a freezing method such as a slow freezing method or a vitrification freezing method. As used herein, the term “slow freezing method” refers to a method of freezing cells by infiltrating the cells with a cryoprotectant and then gradually lowering the temperature. The cooling temperature in the slow freezing method is, for example, 0°C or less, -10°C or less, -20°C or less, -30°C or less, -40°C or less, -50°C or less, -60°C or less, -70°C or less, Or it may be -80°C or lower. As used herein, the term “vitrification freezing method” refers to a method of freezing cells using vitrification. "Vitrification" is a phenomenon in which a liquid does not crystallize but becomes a solid depending on cooling conditions such as cooling rate and pressure, and it is known that water also vitrifies depending on the conditions. The vitrification freezing method is often used when efficient preservation by the slow freezing method is difficult. The cooling temperature in the vitrification freezing method is, for example, -100°C or less, -110°C or less, -120°C or less, -130°C or less, -140°C or less, -150°C or less, -160°C or less, -170°C or less , -180°C or lower, -190°C or lower, or -200°C or lower. The cooling rate is not limited as long as it does not significantly impair the cell viability after freezing and thawing, and can be appropriately selected according to a freezing method such as slow freezing or vitrification. For the slow freezing method, for example, -1°C/min to -0.1°C/min, -0.8°C/min to -0.2°C/min, or -0.6°C/min to -0.3°C/min, preferably -0.5°C /min to -0.4°C/min. Such a cooling rate can be achieved by housing a cryopreservation container such as a cryotube in a further freezing treatment container and subjecting it to cooling with a deep freezer or liquid nitrogen. -5°C/min to -250°C/min, -10°C/min to -240°C/min, -15°C/min to -220°C/min, -20°C/min or more for the vitrification freezing method -200°C/min, -50°C/min to -180°C/min, -100°C/min to -160°C/min, or -120°C/min to -150°C/min, liquid nitrogen Rapid freezing using, etc. is preferably used. Cells after the freezing step can be maintained in a frozen state until the thawing step described below.
(解凍工程)
本態様の方法は、凍結した細胞を解凍する解凍工程を必須工程として含む。
本工程において細胞を解凍する方法は限定せず、任意の解凍方法により行うことができる。具体的には、凍結した細胞を解凍可能な温度以上の温度に供することによって細胞を解凍することができる。そのような温度の例としては、2℃~60℃、4℃~50℃、10℃~45℃、20℃~42℃、30℃~40℃、35℃~38℃、又は36℃~37℃が挙げられ、例えば約37℃であってもよい。また、解凍時間は、細胞の生存率や増殖能を低下させないものであれば限定せず、4分以内、3分以内、2分以内、又は1分以内であり、好ましくは30秒以内又は20秒以内であり得る。
(Thawing process)
The method of this embodiment includes a thawing step of thawing frozen cells as an essential step.
The method for thawing the cells in this step is not limited, and any thawing method can be used. Specifically, frozen cells can be thawed by subjecting them to a temperature at which they can be thawed or higher. Examples of such temperatures are 2°C to 60°C, 4°C to 50°C, 10°C to 45°C, 20°C to 42°C, 30°C to 40°C, 35°C to 38°C, or 36°C to 37°C. °C may be mentioned, for example about 37°C. In addition, the thawing time is not limited as long as it does not reduce the cell viability or proliferation ability, and is within 4 minutes, 3 minutes, 2 minutes, or 1 minute, preferably 30 seconds or 20 seconds. can be within seconds.
凍結した細胞を解凍可能な温度以上の温度に供する手段は限定せず、クライオチューブ等に入れた状態でウォーターバス、インキュベーター、又は恒温器で温める方法や、クライオチューブ等をヒトの手で温める方法、凍結した細胞を予め目的の温度にした培養液等の液体中に浸漬する方法が例示される。 There are no restrictions on the means for exposing frozen cells to a temperature higher than the temperature at which they can be thawed. A method of warming the cells in a cryotube, etc. in a water bath, incubator, or thermostat, or a method of manually warming the cryotube, etc. , a method of immersing frozen cells in a liquid such as a culture solution that has been brought to a desired temperature in advance.
解凍後の細胞は、後述の増殖工程又は第一培養工程で培養に用いる培地に添加することができるが、必要に応じて細胞を洗浄してもよい。洗浄液には、必要に応じて糖等の添加物を加えたバッファーや生理食塩水、及び/又は後述の増殖工程又は第一培養工程で培養に用いる培地等を使用することができる。 The thawed cells can be added to the medium used for culture in the growth step or first culture step described below, but the cells may be washed as necessary. As the washing liquid, a buffer or physiological saline solution containing additives such as sugars as necessary, and/or a medium used for culture in the later-described growth step or first culture step, or the like can be used.
(増殖工程)
本態様の方法は、細胞を血清若しくは血漿含有培地で培養して増殖させる増殖工程を選択工程として含む。
(Proliferation step)
The method of this embodiment includes, as a selection step, a growth step in which the cells are cultured and grown in a serum- or plasma-containing medium.
増殖工程の培養条件は限定されず、例えば、培養温度は約30℃~約40℃、CO2濃度は約2%~約10%であってよく、接着培養であっても懸濁培養であってもよい。 The culture conditions in the growth step are not limited, and for example, the culture temperature may be about 30° C. to about 40° C., and the CO 2 concentration may be about 2% to about 10%. may
また、増殖の時間は、特に限定しない。例えば、増殖工程は、数時間~数日、1日~4日、2日~3日、又は2日行ってもよい。 Also, the growth time is not particularly limited. For example, the growth step may occur for hours to days, 1 to 4 days, 2 to 3 days, or 2 days.
本工程では、増殖後に培養上清をデカンテーション及びアスピレーション等の通常の方法で除去することができる。 In this step, the culture supernatant can be removed by ordinary methods such as decantation and aspiration after growth.
(繰り返し工程)
本態様の方法は、上述の増殖工程を1回以上繰り返す繰り返し工程を選択工程として含む。
(Repeated process)
The method of this embodiment includes, as a selection step, a repeating step of repeating the growth step described above one or more times.
繰り返し工程において、増殖工程を繰り返す回数は1回以上であり、例えば、2回以上、3回以上、4回以上、若しくは5回以上、及び/又は5回以下、4回以下、3回以下、若しくは2回以下であり、好ましくは1~3回、1~2回、又は1回あってもよい。 In the repeating step, the number of times the growing step is repeated is 1 or more times, for example, 2 times or more, 3 times or more, 4 times or more, or 5 times or more, and/or 5 times or less, 4 times or less, 3 times or less, Or 2 times or less, preferably 1 to 3 times, 1 to 2 times, or 1 time.
本工程と本工程に含まれない増殖工程とを合わせた培養時間は、4時間以上、8時間以上、又は24時間以上であってよく、及び/又は120時間以下、96時間以下、72時間以下、60時間以下、54時間以下、又は48時間以下、好ましくは8時間~96時間又は24時間~72時間であってよい。 The total culture time for this step and the growth step not included in this step may be 4 hours or more, 8 hours or more, or 24 hours or more, and/or 120 hours or less, 96 hours or less, or 72 hours or less. , 60 hours or less, 54 hours or less, or 48 hours or less, preferably 8 hours to 96 hours or 24 hours to 72 hours.
(第一培養工程)
本態様の方法は、細胞を第一の培地で培養する、第一培養工程を必須工程として含む。本明細書において「第一の培地」とは、第一培養工程で細胞を培養するために用いる培地をいう。第一の培地は、基本培地又は無血清培地である。
(First culture step)
The method of this aspect includes, as an essential step, a first culturing step of culturing cells in a first medium. As used herein, the term "first medium" refers to a medium used for culturing cells in the first culture step. The first medium is a basal medium or a serum-free medium.
一実施形態において、第一の培地はタンパク質を含む。第一の培地に含まれるタンパク質により、本工程において細胞の増殖、接着、及び細胞機能誘導(分化誘導若しくは未分化維持、細胞老化抑制、又は特定遺伝子発現の誘導等を含む)等を促進することができる。第一の培地が含むことができるタンパク質は、ヒトタンパク質又は非ヒトタンパク質のいずれであってもよく、そのさらなる例は、上述の前培養工程の説明に準じるものとする。それ故、ここでの詳細な説明は省略する。 In one embodiment, the first medium contains protein. The protein contained in the first medium promotes cell proliferation, adhesion, and cell function induction (including induction of differentiation or maintenance of undifferentiation, suppression of cell senescence, induction of specific gene expression, etc.) in this step. can be done. The proteins that the first medium may contain may be either human proteins or non-human proteins, further examples of which follow the description of the pre-culturing step above. Therefore, detailed description is omitted here.
別の実施形態において、第一の培地はタンパク質を含まない。
第一培養工程の培養条件は限定されず、例えば、培養温度は約30℃~約40℃、CO2濃度は約2%~約10%であってよく、接着培養であっても懸濁培養であってもよい。また、第一培養工程の時間は、特に限定しない。例えば、第一培養工程は、数時間~数週間、数か月、又は数日行ってもよい。第一培養工程のより具体的な培養時間は、30分以上、1時間以上、1時間30分以上、2時間以上、4時間以上、8時間以上、又は24時間以上であってよく、及び/又は120時間以下、96時間以下、72時間以下、60時間以下、54時間以下、又は48時間以下であってよい。例えば、第一培養工程の時間は、12時間~120時間、又は約48時間とすることができる。細胞が間葉系幹細胞である場合、第一培養工程の好ましい培養時間は12時間~48時間である。
In another embodiment, the first medium does not contain protein.
The culture conditions in the first culture step are not limited. may be Moreover, the time of the first culture step is not particularly limited. For example, the first culturing step may take hours to weeks, months, or days. A more specific culture time of the first culture step may be 30 minutes or longer, 1 hour or longer, 1 hour and 30 minutes or longer, 2 hours or longer, 4 hours or longer, 8 hours or longer, or 24 hours or longer, and/ or 120 hours or less, 96 hours or less, 72 hours or less, 60 hours or less, 54 hours or less, or 48 hours or less. For example, the duration of the first culturing step can be 12 hours to 120 hours, or about 48 hours. When the cells are mesenchymal stem cells, the preferred culture time for the first culture step is 12 hours to 48 hours.
(除去工程)
本態様の方法は、第一培養工程後に得られる培養上清を除去する除去工程を必須工程として含む。
(Removal process)
The method of this aspect includes, as an essential step, a removing step of removing the culture supernatant obtained after the first culturing step.
培養上清の除去はデカンテーション及びアスピレーション等の通常の方法で行うことができる。ここで、第一の培地の成分(例えば、ヒトタンパク質又は非ヒトタンパク質等)が第二培養工程で用いる第二の培地に混入しないように、第一培養工程の培養上清を完全に除去することが好ましい。 Removal of the culture supernatant can be carried out by ordinary methods such as decantation and aspiration. Here, the culture supernatant in the first culturing step is completely removed so that the components of the first medium (e.g., human protein or non-human protein, etc.) do not contaminate the second medium used in the second culturing step. is preferred.
(洗浄工程)
本態様の方法は、除去工程後、第二培養工程前に細胞を洗浄する洗浄工程を選択工程として含む。洗浄工程において用いる洗浄液は、細胞の生存性に過度な影響を与えない限り限定されず、例えばバッファー、生理食塩水、又は後述する第二の培地や、これらの溶液に糖等の添加物を加えたものであってよい。洗浄の回数は、例えば1回以上であり、例えば1回、2回、3回、又は4回とすることができる。
(Washing process)
The method of this embodiment includes a washing step of washing the cells after the removing step and before the second culturing step as a selection step. The washing solution used in the washing step is not limited as long as it does not excessively affect the viability of the cells. can be anything. The number of washings is, for example, one or more times, and can be, for example, one, two, three, or four times.
(第二培養工程)
本態様の方法は、除去工程後の細胞をさらに第二の培地で培養する第二培養工程を必須工程として含む。本明細書において「第二の培地」とは、第二培養工程で細胞を培養するために用いる培地をいう。第二の培地は、タンパク質を含まない培地(以下、「タンパク質不含培地」という)である。
(Second culture step)
The method of this aspect includes, as an essential step, a second culture step of further culturing the cells after the removal step in a second medium. As used herein, the term "second medium" refers to a medium used for culturing cells in the second culture step. The second medium is a protein-free medium (hereinafter referred to as "protein-free medium").
本明細書において、「タンパク質不含培地」とは、タンパク質を含まない、又は実質的に含まない培地を意味する。タンパク質不含培地の例として、タンパク質を含まない無血清培地、及びタンパク質を含まない基本培地が挙げられる。 As used herein, "protein-free medium" means a medium that does not contain or substantially does not contain protein. Examples of protein-free media include protein-free serum-free media and protein-free basal media.
第二培養工程の培養条件は限定されず、例えば、培養温度は約30℃~約40℃、CO2濃度は約2%~約10%であってよく、接着培養であっても懸濁培養であってもよい。 The culture conditions of the second culture step are not limited. may be
第二培養工程の時間は、後述する回収工程後に得られる目的の産物に応じて適宜選択することができる。第二培養工程は、例えば12時間以上、18時間以上、24時間以上、36時間以上、又は42時間以上であってよく、また120時間以下、96時間以下、72時間以下、60時間以下、又は54時間以下であってよい。例えば、第二培養工程の時間は、12時間~120時間、24時間~72時間、42時間~54時間又は約48時間とすることができる。 The time for the second culture step can be appropriately selected according to the target product obtained after the recovery step described below. The second culturing step may be, for example, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, or 42 hours or more, or 120 hours or less, 96 hours or less, 72 hours or less, 60 hours or less, or May be 54 hours or less. For example, the duration of the second culturing step can be 12 hours to 120 hours, 24 hours to 72 hours, 42 hours to 54 hours, or about 48 hours.
本明細書において、「目的の産物」とは、本発明の方法によって第二培養工程後に得られる培養上清に含まれる産物を指し、治療用タンパク質、ホルモン等の低分子、エクソソーム、ウイルス、又はこれらの組み合わせが挙げられる。治療用タンパク質は、限定するものではないが、例えば酵素、ペプチドホルモン等のホルモン、血液凝固因子、サイトカイン、増殖因子、又は抗体若しくは抗体断片の他、プログラニュリンやセマフォリン等が例示される。増殖因子の例としては、肝細胞増殖因子(HGF)、血管内皮細胞増殖因子(VEGF)、上皮増殖因子(EGF)、線維芽細胞増殖因子(FGF)、神経成長因子(NGF)、脳由来神経栄養因子(BDGF)、胎盤成長因子(PLGF)、骨形成因子(BMP)等が挙げられる。 As used herein, the term "product of interest" refers to a product contained in the culture supernatant obtained after the second culture step by the method of the present invention, and includes therapeutic proteins, small molecules such as hormones, exosomes, viruses, or Combinations of these are included. Examples of therapeutic proteins include, but are not limited to, enzymes, hormones such as peptide hormones, blood coagulation factors, cytokines, growth factors, antibodies or antibody fragments, as well as progranulin and semaphorin. Examples of growth factors include hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived nerve trophic factor (BDGF), placental growth factor (PLGF), bone morphogenetic protein (BMP) and the like.
(回収工程)
本態様の方法は、第二培養工程後に得られる培養上清を回収する回収工程を必須工程として含む。回収方法は特に限定しない。回収された培養上清は、遠心分離及び/又は膜ろ過等により、細胞や細胞に由来するデブリを除去してもよい。
(Recovery process)
The method of this aspect includes, as an essential step, a recovery step of recovering the culture supernatant obtained after the second culture step. The collection method is not particularly limited. Cells and cell-derived debris may be removed from the collected culture supernatant by centrifugation and/or membrane filtration.
(精製工程)
本態様の方法は、回収工程後に得られた培養上清から、目的の産物をさらに精製する工程を選択工程として含む。例えば、目的の産物がタンパク質であれば、常法により、ゲルろ過クロマトグラフィー、イオン交換カラムクロマトグラフィー、アフィニティークロマトグラフィー、逆相カラムクロマトグラフィー、HPLC等のクロマトグラフィー、硫安分画、限外ろ過、及び免疫吸着法等により、また、目的の産物がエクソソームであれば、超遠心分離、免疫沈降、限外濾過、高分子沈殿法、フィールドフローフラクショネーション法、及び遠心フィールドフローフラクショネーション法等により精製することができる。
(Refining process)
The method of this embodiment includes, as a selection step, a step of further purifying the target product from the culture supernatant obtained after the collection step. For example, if the target product is a protein, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reversed-phase column chromatography, chromatography such as HPLC, ammonium sulfate fractionation, ultrafiltration, And by immunoadsorption method, etc., and if the target product is exosomes, ultracentrifugation, immunoprecipitation, ultrafiltration, polymer precipitation method, field flow fractionation method, centrifugal field flow fractionation method, etc. can be purified by
(効果)
本態様の方法によれば、高濃度の生物活性物質を含む培養上清を生産することができる。本態様の方法では、細胞を薬物やホルモン等で刺激することなく高濃度の生物活性物質を含む培養上清を生産することができるため、培養上清を治療用途に用いる場合等に安全性を簡便に担保することができることも利点である。それ故、ヒトに対して適用した場合に、アレルギー反応等の副作用を生ずるリスクが低く、例えばヒトにおいて医療用途、化粧品用途、健康食品用途の少なくとも一つ以上に用いることができる。
(effect)
According to the method of this embodiment, a culture supernatant containing a high concentration of biologically active substances can be produced. The method of this embodiment can produce a culture supernatant containing a biologically active substance at a high concentration without stimulating cells with drugs, hormones, etc. Therefore, the culture supernatant can be safely used for therapeutic purposes. Another advantage is that it can be secured easily. Therefore, when applied to humans, the risk of side effects such as allergic reactions is low, and for example, it can be used for at least one or more of medical, cosmetic, and health food applications in humans.
<培養上清>
一態様において、本発明は、本明細書に記載される方法により生産される培養上清に関する。本発明の培養上清は、培養を行う細胞の種類や培養条件に応じて、様々な物質を含み得る。例えば、培養上清は、遺伝子組換えを行っていない細胞が分泌する天然のタンパク質、及び/又はエクソソームを含んでもよいし、遺伝子組換えを行った細胞が分泌する組換えタンパク質、及び/又はエクソソームを含んでもよい。培養上清に含まれるタンパク質の例として、治療用タンパク質、例えば酵素、ホルモン、血液凝固因子、サイトカイン、増殖因子、抗体等、又はこれらの組み合わせが挙げられる。
<Culture supernatant>
In one aspect, the invention relates to culture supernatants produced by the methods described herein. The culture supernatant of the present invention may contain various substances depending on the type of cells to be cultured and the culture conditions. For example, the culture supernatant may contain natural proteins secreted by non-genetically modified cells, and / or exosomes, or recombinant proteins secreted by genetically modified cells, and / or exosomes may include Examples of proteins contained in the culture supernatant include therapeutic proteins such as enzymes, hormones, blood clotting factors, cytokines, growth factors, antibodies, etc., or combinations thereof.
本発明の培養上清は、そのまま用いてもよいし、凍結乾燥等によって、必要に応じて製剤化を行って用いてもよい。製剤化は、医薬的に許容される担体や添加物を用いて、常法に従って行うことができる(例えば、Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton,米国を参照されたい)。例えば、本発明の培養上清を含む組成物、並びに本発明の培養上清及び医薬的に許容される担体や添加物を含む組成物(例えば医薬組成物)も提供される。 The culture supernatant of the present invention may be used as it is, or may be formulated by freeze-drying or the like, if necessary. Formulation can be carried out according to conventional methods using pharmaceutically acceptable carriers and additives (see, for example, Remington's Pharmaceutical Sciences, latest edition, Mark Publishing Company, Easton, USA). For example, compositions containing the culture supernatant of the present invention and compositions (eg, pharmaceutical compositions) containing the culture supernatant of the present invention and pharmaceutically acceptable carriers and additives are also provided.
医薬的に許容される担体及び添加物の例としては、限定されるものではないが、水、医薬的に許容される有機溶剤、例えばポリエチレングリコール、ジグリセリン、グリセリン、プロピレングリコール、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、メチルセルロース、エチルセルロース、及びキサンタンガム等の増粘剤;ワセリン及びパラフィン等の脂質;マンニトール、ソルビトール、ラクトース等の糖アルコール、及び糖類;Tween 80及びTween 20等の界面活性剤等が挙げられ、添加物は単独で又は適宜組み合わせて用いられる。
Examples of pharmaceutically acceptable carriers and additives include, but are not limited to, water, pharmaceutically acceptable organic solvents such as polyethylene glycol, diglycerin, glycerin, propylene glycol, polyvinyl alcohol, polyvinyl Thickeners such as pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, water-soluble dextran, sodium carboxymethyl starch, methylcellulose, ethylcellulose, and xanthan gum; lipids such as petrolatum and paraffin; sugar alcohols such as mannitol, sorbitol, and lactose, and sugars. surfactants such as Tween 80 and
本態様の培養上清によれば、本態様の培養上清を被験体に投与する工程を含む、疾患を治療する方法、被験体の損傷を治癒する方法、及び被験体において免疫を抑制する方法が提供される。疾患の例として、肝炎、肝硬変、神経再生、脊髄損傷、変形性関節症、慢性疼痛、脳梗塞、アルツハイマー型認知症、多発性硬化症、パーキンソン病、筋萎縮性側索硬化症、低酸素性虚血性脳症、末梢神経障害、糖尿病、糖尿病性神経障害、糖尿病性腎症、虚血性心疾患、心不全、間質性肺炎、骨欠損、顎骨壊死、歯周病、アレルギー性皮膚炎、脱毛症、末梢動脈疾患、クローン病、潰瘍性大腸炎、関節リウマチ、膠原病、慢性腎不全、腹圧性尿失禁、うつ病、慢性頭痛、不眠症、眼精疲労、褥瘡、重度熱傷、腹圧性尿失禁、骨粗しょう症、難治性骨折、更年期障害、浮腫、及びギランバレー症候群が挙げられる。 According to the culture supernatant of this aspect, a method of treating a disease, a method of healing an injury in a subject, and a method of suppressing immunity in a subject, comprising the step of administering the culture supernatant of this aspect to a subject. is provided. Examples of diseases include hepatitis, liver cirrhosis, nerve regeneration, spinal cord injury, osteoarthritis, chronic pain, cerebral infarction, Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, hypoxia Ischemic encephalopathy, peripheral neuropathy, diabetes, diabetic neuropathy, diabetic nephropathy, ischemic heart disease, heart failure, interstitial pneumonia, bone defect, osteonecrosis of the jaw, periodontal disease, allergic dermatitis, alopecia, Peripheral arterial disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, collagen disease, chronic renal failure, stress urinary incontinence, depression, chronic headache, insomnia, eye strain, pressure ulcer, severe burns, stress urinary incontinence, Osteoporosis, refractory fractures, menopause, edema, and Guillain-Barré syndrome.
また、本態様の培養上清によれば、疾患の治療において使用するための培養上清、損傷治癒において使用するための培養上清、及び免疫抑制における使用のための培養上清が提供される。 In addition, according to the culture supernatant of this aspect, culture supernatants for use in disease treatment, culture supernatants for use in wound healing, and culture supernatants for use in immunosuppression are provided. .
疾患の治療、損傷治癒、又は免疫抑制のための医薬の製造における、本態様の培養上清の使用もまた提供される。 Also provided is the use of the culture supernatant of this aspect in the manufacture of a medicament for the treatment of disease, wound healing, or immunosuppression.
<サイトカイン及び/又はエクソソームの含有量を増加させる方法>
一態様において、本発明は、培養上清においてサイトカイン、及び/又はエクソソームの含有量を増加させる方法に関する。本態様の方法は、解凍工程、第一培養工程、除去工程、第二培養工程、及び回収工程を必須工程として含む。また、選択工程として、増殖工程、繰り返し工程、洗浄工程、及び精製工程等の他の工程を含んでもよい。さらなる選択工程として、解凍工程の前に、前培養工程、置換工程、及び凍結工程を含んでもよい。本発明の方法を構成する各工程のうち、回収工程以外の工程は、上述の培養上清を生産する方法に準じるものとする。それ故、これらの工程についての説明は省略し、ここでは本態様の方法における回収工程についてのみ以下説明する。
<Method for increasing the content of cytokines and / or exosomes>
In one aspect, the present invention relates to a method of increasing the content of cytokines and/or exosomes in culture supernatants. The method of this embodiment includes, as essential steps, a thawing step, a first culturing step, a removing step, a second culturing step, and a collecting step. Selection steps may also include other steps such as growth steps, repetition steps, washing steps, and purification steps. Additional selection steps may include a pre-incubation step, a replacement step, and a freezing step prior to the thawing step. Among the steps constituting the method of the present invention, the steps other than the recovery step conform to the above-described method for producing the culture supernatant. Therefore, description of these steps will be omitted, and only the recovery step in the method of this embodiment will be described below.
(回収工程)
本態様の方法において、回収工程は、第二培養工程後に得られるサイトカイン、及び/又はエクソソームを含む培養上清を回収する工程である。
(Recovery process)
In the method of this aspect, the recovery step is a step of recovering the culture supernatant containing cytokines and/or exosomes obtained after the second culture step.
本工程で回収したサイトカイン、及び/又はエクソソームを含む培養上清は、遠心分離及び/又は膜ろ過等により、細胞や細胞に由来するデブリを除去してもよい。また、培養上清中のサイトカイン、及び/又はエクソソームを常法により精製することもできる。例えば、サイトカインをゲルろ過クロマトグラフィー、イオン交換カラムクロマトグラフィー、アフィニティークロマトグラフィー、逆相カラムクロマトグラフィー、HPLC等のクロマトグラフィー、硫安分画、限外ろ過、及び免疫吸着法等により生成することができる。また、エクソソームを超遠心分離、免疫沈降、限外濾過、高分子沈殿法、フィールドフローフラクショネーション法、遠心フィールドフローフラクショネーション法等により精製することができる。 The culture supernatant containing cytokines and/or exosomes collected in this step may be subjected to centrifugation and/or membrane filtration to remove cells and cell-derived debris. In addition, cytokines in the culture supernatant and / or exosomes can be purified by a conventional method. For example, cytokines can be produced by gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, chromatography such as HPLC, ammonium sulfate fractionation, ultrafiltration, immunoadsorption, and the like. . In addition, exosomes can be purified by ultracentrifugation, immunoprecipitation, ultrafiltration, polymer precipitation, field flow fractionation, centrifugal field flow fractionation, and the like.
以下、実施例を参照して本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
<実施例1:解凍した細胞を用いた培養上清の生産>
(目的)
解凍後の細胞を用いて培養上清を生産する際に生物活性物質の含有量が向上し得る条件について検討する。
<Example 1: Production of culture supernatant using thawed cells>
(the purpose)
The conditions under which the content of the bioactive substance can be improved when the culture supernatant is produced using the thawed cells are examined.
(方法)
本実施例では、凍結及び解凍を含む方法(方法A~C)、及び凍結及び解凍を含まない方法(対照1)の各方法により培養上清を生産した。各々の方法に含まれる各工程を以下で説明し、その概要を図3に図示する。
(Method)
In this example, culture supernatants were produced by methods including freezing and thawing (Methods A to C) and methods not including freezing and thawing (Control 1). Each step involved in each method is described below and outlined graphically in FIG.
方法A:方法Aは、後述する(1)前培養、(2)細胞の凍結、(3)細胞の解凍、(4)無血清培地及び/又は血清含有培地での培養の(4-1)方法A、(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む(図3、「方法A」)。 Method A: Method A is (4-1) of (1) pre-culture, (2) freezing of cells, (3) thawing of cells, (4) culture in serum-free medium and/or serum-containing medium, which will be described later. Method A includes the steps of (5) culturing in basal medium and (6) collecting the culture supernatant (Fig. 3, "Method A").
方法B:方法Bは、後述する(1)前培養、(2)細胞の凍結、(3)細胞の解凍、(4)無血清培地及び/又は血清含有培地での培養の(4-2)方法B、(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む(図3、「方法B」)。 Method B: Method B is (4-2) of (1) preculture, (2) freezing of cells, (3) thawing of cells, (4) culture in serum-free medium and/or serum-containing medium, which will be described later. Method B includes the steps of (5) culturing in basal medium and (6) collecting the culture supernatant (Fig. 3, "Method B").
方法C:方法Cは、後述する(1)前培養、(2)細胞の凍結、(3)細胞の解凍、(4)無血清培地及び/又は血清含有培地での培養の(4-3)方法C、(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む(図3、「方法C」)。 Method C: Method C includes (1) preculture, (2) cell freezing, (3) cell thawing, (4) culture in serum-free medium and/or serum-containing medium (4-3), which will be described later. Method C includes the steps of (5) culturing in basal medium and (6) collecting the culture supernatant (Fig. 3, "Method C").
凍結及び解凍を含まない方法(対照1):対照として行った方法には、後述する(2)細胞の凍結、及び(3)細胞の解凍が含まれない。この方法(対照1)は、後述する(1)前培養の後に無血清培地で培養を行い、次いで(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む(図3、「対照1」)。 Method without Freezing and Thawing (Control 1): The method performed as a control does not include (2) cell freezing and (3) cell thawing, as described below. This method (control 1) includes the steps of (1) culturing in a serum-free medium after preculture, then (5) culturing in a basal medium, and (6) recovering the culture supernatant ( Fig. 3, "Control 1").
以下、各工程について説明する。
(1)前培養
皮下脂肪由来間葉系幹細胞(Mesenchymal Stem Cells: MSC)を血清含有培地で培養した。血清含有培地として、FBSを添加したPRIME-XV MSC Expansion XSFM(Irvine Scientific、#91149-1L)を使用した。
Each step will be described below.
(1) Preculture Subcutaneous adipose-derived mesenchymal stem cells (MSCs) were cultured in a serum-containing medium. PRIME-XV MSC Expansion XSFM (Irvine Scientific, #91149-1L) supplemented with FBS was used as serum-containing medium.
培養用容器内での間葉系幹細胞の細胞密度が80%程度に達した後に、細胞を金属イオン不含リン酸緩衝生理食塩水(Phosphate Buffer Saline(-):PBS(-))で洗浄した。次いで細胞剥離液であるアクターゼ(Innovative Cell Technologies、#AT104-500)により細胞を培養用容器から剥離した。培地を加えて細胞を遠心チューブに移した。 After the cell density of mesenchymal stem cells in the culture vessel reached about 80%, the cells were washed with metal ion-free phosphate buffer saline (PBS(-)). . Next, the cells were detached from the culture vessel with a cell detachment solution, Actase (Innovative Cell Technologies, #AT104-500). Medium was added and the cells were transferred to a centrifuge tube.
低速遠心操作(1,200rpm、5分)により細胞を上記の溶液から分離し、上澄み液を廃棄した。PBS(-)により細胞を再浮遊させることで洗浄した。細胞浮遊液を一部分取し、細胞数を計測した。低速遠心操作により、再び細胞を分離した。 Cells were separated from the above solution by low speed centrifugation (1,200 rpm, 5 minutes) and the supernatant was discarded. The cells were washed by resuspending them with PBS(-). A portion of the cell suspension was taken and the number of cells was counted. Cells were separated again by low speed centrifugation.
(2)細胞の凍結
細胞密度が1~3×106細胞/mLとなるようにバンバンカー(GCリンフォテック、#CS-02-001)を加えて、1.5mLのクライオチューブに1mL分注した。
(2) Cell freezing Add Bambanker (GC Lymphotech, #CS-02-001) so that the cell density is 1-3×10 6 cells/mL, and dispense 1 mL into 1.5 mL cryotubes. bottom.
分注したクライオチューブを4℃で予冷しておいたBICELL(登録商標)(日本フリーザー)製の凍結容器に入れ、同容器を-80℃の冷凍庫に3時間以上静置し、-0.5℃/分~-0.4℃/分の冷却速度で細胞を凍結した。凍結された細胞は、解凍するまで-80℃又は液体窒素を用いた保存タンクで保存した。 Place the dispensed cryotube in a BICELL (registered trademark) (Nippon Freezer) freezing container that has been precooled at 4°C, and place the container in a freezer at -80°C for 3 hours or more. Cells were frozen at a cooling rate of min to -0.4°C/min. Frozen cells were stored at −80° C. or in storage tanks with liquid nitrogen until thawed.
(3)細胞の解凍
上記(1)で凍結保存した細胞を37℃で素早く解凍した。解凍された1mLの細胞を遠心チューブに移し、10倍量のDulbecco Minimum Essential Media(DMEM)をゆっくり加えた。
(3) Cell Thawing The cells cryopreserved in (1) above were quickly thawed at 37°C. 1 mL of thawed cells were transferred to a centrifugation tube, and 10 volumes of Dulbecco Minimum Essential Media (DMEM) were slowly added.
低速遠心操作により、細胞を上記溶液から分離した。PBS(-)を加え、細胞を浮遊させた。細胞浮遊液を一部分取し、細胞数を計測した。
低速遠心操作により、細胞を分離した。
Cells were separated from the solution by low speed centrifugation. PBS(-) was added to float the cells. A portion of the cell suspension was taken and the number of cells was counted.
Cells were separated by low speed centrifugation.
(4)無血清培地及び/又は血清含有培地での培養
上記(3)で分離した細胞を培養した。培養方法は方法A、方法B、及び方法Cで異なり、以下の(4-1)~(4-3)の各々で具体的に説明する。
(4) Culture in serum-free medium and/or serum-containing medium The cells separated in (3) above were cultured. The culturing method differs between Method A, Method B, and Method C, and will be specifically described in each of (4-1) to (4-3) below.
(4-1)方法A
上記(3)で分離した細胞を無血清培地に再懸濁した。無血清培地として、FBS無添加のPRIME-XV MSC Expansion XSFMを用いた。次いで、同じ無血清培地を入れた培養容器に、約2.7×104細胞/cm2となるように細胞を播種した。
(4-1) Method A
The cells separated in (3) above were resuspended in serum-free medium. PRIME-XV MSC Expansion XSFM without FBS was used as a serum-free medium. Cells were then seeded at approximately 2.7×10 4 cells/cm 2 in culture vessels containing the same serum-free medium.
培養開始から48時間後、培養用容器内での細胞密度が95%程度に達した時点で培地を除去した。 After 48 hours from the start of culture, the medium was removed when the cell density in the culture vessel reached about 95%.
(4-2)方法B
上記(3)で分離した細胞を血清含有培地に再懸濁した。血清含有培地として、FBSを添加したPRIME-XV MSC Expansion XSFMを用いた。同じ血清含有培地を入れた培養容器に、約0.8~1.3×104細胞/cm2となるように細胞を播種した。
(4-2) Method B
The cells separated in (3) above were resuspended in serum-containing medium. PRIME-XV MSC Expansion XSFM supplemented with FBS was used as serum-containing medium. Cells were seeded at approximately 0.8-1.3×10 4 cells/cm 2 in culture vessels containing the same serum-containing medium.
培養開始から約48時間後、培養用容器内での細胞密度が80%程度に達した時点で細胞をPBS(-)で洗浄した。次いで、アクターゼにより細胞を培養用容器から剥離し、培地を加えて細胞を遠心チューブに移した。 About 48 hours after the initiation of culture, when the cell density in the culture vessel reached about 80%, the cells were washed with PBS(-). Then, the cells were detached from the culture vessel by actase, medium was added, and the cells were transferred to a centrifugation tube.
低速遠心操作により細胞を上記溶液から分離し、上澄み液を廃棄した。PBS(-)により細胞を再浮遊させることで洗浄した。細胞浮遊液を一部分取し、細胞数を計測した。低速遠心操作により、再び細胞を分離した。 Cells were separated from the solution by low speed centrifugation and the supernatant was discarded. The cells were washed by resuspending them with PBS(-). A portion of the cell suspension was taken and the number of cells was counted. Cells were separated again by low speed centrifugation.
次いで、分離した細胞を無血清培地に再懸濁した。無血清培地として、FBS無添加のPRIME-XV MSC Expansion XSFMを用いた。1.3×104細胞/cm2となるように、同じ無血清培地を入れた培養容器に細胞を播種した。 Detached cells were then resuspended in serum-free medium. PRIME-XV MSC Expansion XSFM without FBS was used as a serum-free medium. Cells were seeded in culture vessels containing the same serum-free medium at 1.3×10 4 cells/cm 2 .
培養開始から48時間後、培養用容器内での細胞密度が95%程度に達した時点で培地を除去した。 After 48 hours from the start of culture, the medium was removed when the cell density in the culture vessel reached about 95%.
(4-3)方法C
上記(3)で分離した細胞を血清含有培地に再懸濁した。血清含有培地として、FBSを添加したPRIME-XV MSC Expansion XSFMを用いた。同じ血清含有培地を入れた培養容器に、約0.8~1.3×104細胞/cm2となるように細胞を播種した。
(4-3) Method C
The cells separated in (3) above were resuspended in serum-containing medium. PRIME-XV MSC Expansion XSFM supplemented with FBS was used as serum-containing medium. Cells were seeded at approximately 0.8-1.3×10 4 cells/cm 2 in culture vessels containing the same serum-containing medium.
培養開始から約48時間後、培養用容器内での細胞密度が80%程度に達した時点で細胞をPBS(-)で洗浄した。次いで、アクターゼにより細胞を培養用容器から剥離し、培地を加えて細胞を遠心チューブに移した。 About 48 hours after the initiation of culture, when the cell density in the culture vessel reached about 80%, the cells were washed with PBS(-). Then, the cells were detached from the culture vessel by actase, medium was added, and the cells were transferred to a centrifugation tube.
低速遠心操作により細胞を上記溶液から分離し、上澄み液を廃棄した。PBS(-)により細胞を再浮遊させることで洗浄した。細胞浮遊液を一部分取し、細胞数を計測した。低速遠心操作により、細胞を再分離した。 Cells were separated from the solution by low speed centrifugation and the supernatant was discarded. The cells were washed by resuspending them with PBS(-). A portion of the cell suspension was taken and the number of cells was counted. Cells were re-separated by low speed centrifugation.
次いで、再分離した細胞を血清含有培地に再懸濁し、上と同様の方法により、血清含有培地での約48時間培養から再分離までを繰り返した。 The re-isolated cells were then resuspended in serum-containing medium, and the same procedure as above was repeated from culturing in serum-containing medium for about 48 hours to re-isolation.
その後、細胞を無血清培地に再懸濁した。無血清培地として、FBS無添加のPRIME-XV MSC Expansion XSFMを用いた。1.3×104細胞/cm2となるように、同じ無血清培地を入れた培養容器に細胞を播種した。 Cells were then resuspended in serum-free medium. PRIME-XV MSC Expansion XSFM without FBS was used as a serum-free medium. Cells were seeded in culture vessels containing the same serum-free medium at 1.3×10 4 cells/cm 2 .
培養開始から48時間後、培養用容器内での細胞密度が95%程度に達した時点で培地を除去した。 After 48 hours from the start of culture, the medium was removed when the cell density in the culture vessel reached about 95%.
(5)基本培地での培養
培養液を除去したフラスコ内の細胞を、段階的に増やした液量(10mL、20mL、30mL、及び40mL)のPBS(-)により計4回洗浄した。基本培地をそれぞれのフラスコに加え、48時間培養した。なお、基本培地としてFBSを含まないDMEM培地を使用した。
(5) Cultivation in basal medium The cells in the flask from which the culture medium was removed were washed with stepwise increased volumes of PBS(-) four times (10 mL, 20 mL, 30 mL, and 40 mL). Basal medium was added to each flask and cultured for 48 hours. DMEM medium containing no FBS was used as the basal medium.
(6)培養上清の回収と生物活性物質含有量の評価
基本培地で48時間培養した後、培養液を回収して高速遠心操作によりデブリを除去し、培養上清を得た。一部の培養上清を用いて、HGF、VEGF、プログラニュリン含量を、サンドウィッチELISA法により定量した。HGFはQuantikine ELISA Human HGF Immunoassay(R&D Systems、#DHG00B)、VEGFはQuantikine ELISA Human VEGF Immunoassay(R&D Systems、#DVE00)、プログラニュリンはProgranulin (human) ELISA kit(AdipoGen Life Science、#AG-45A-0018YEK-KI01)を用いて測定した。また、エクソソーム含有量については、CD63-Capture ヒトエクソソームELISAキット(富士フイルム和光純薬、#290-83601)を用いて測定した。測定方法については、キットに付属の説明書に従って実施した。簡潔に説明すると、キットに従って、必要量の培養上清を96ウェルプレートの各ウェルに添加した。所定のインキュベーション時間の後、培養上清を添加したウェルを付属の洗浄液により4回洗浄した。各キット付属の検出抗体を添加してさらにインキュベーションした後に、ウェルを洗浄した。洗浄後の各ウェルにTMB基質を添加して発色反応を10分又は30分行い、反応停止液により発色反応を停止し、450nmの吸光度を測定した。
(6) Collection of Culture Supernatant and Evaluation of Bioactive Substance Content After culturing in the basal medium for 48 hours, the culture medium was collected and debris was removed by high-speed centrifugation to obtain the culture supernatant. A portion of the culture supernatant was used to quantify HGF, VEGF and progranulin content by sandwich ELISA. HGF is Quantikine ELISA Human HGF Immunoassay (R&D Systems, #DHG00B), VEGF is Quantikine ELISA Human VEGF Immunoassay (R&D Systems, #DVE00), Progranulin is Progranulin (human) ELISA kit (AdipoGen Life Science, #AG-45A- 0018YEK-KI01). In addition, exosome content was measured using a CD63-Capture human exosome ELISA kit (Fujifilm Wako Pure Chemical Industries, #290-83601). The measurement method was carried out according to the instructions attached to the kit. Briefly, the required amount of culture supernatant was added to each well of a 96-well plate according to the kit. After the predetermined incubation time, the wells to which the culture supernatant was added were washed 4 times with the attached washing solution. After adding the detection antibody attached to each kit and further incubating, the wells were washed. A TMB substrate was added to each well after washing, and the coloring reaction was carried out for 10 minutes or 30 minutes.
(結果)
方法A~C、及び対照1の各方法により生産した培養上清において生物活性物質の含有量を測定した結果を図4に示す。
(result)
FIG. 4 shows the results of measuring the content of biologically active substances in the culture supernatants produced by Methods A to C and Control 1.
図4に示す結果から、凍結及び解凍を含まない対照1と比較して、凍結及び解凍を含む方法A~Cでは、培養上清中の生物活性物質の含有量が多いことが示された。また、方法A~Cを比較した結果、解凍後の継代数が少ないほど培養上清中の生物活性物質の含有量が多いことが示された。特に、方法B及びC並びに対照1の各方法と比較して、方法Aによって生産した培養上清において生物活性物質の含有量が大幅に増加した。具体的にはHGF、VEGF、プログラニュリン、及びエクソソームのいずれの含有量も、方法Aによって生産した細胞上清で最大となった。 The results shown in FIG. 4 indicate that methods A to C with freezing and thawing resulted in higher bioactive substance content in the culture supernatant compared to Control 1 without freezing and thawing. Moreover, as a result of comparing Methods A to C, it was shown that the lower the number of passages after thawing, the higher the content of biologically active substances in the culture supernatant. In particular, there was a significant increase in the content of bioactive substances in the culture supernatant produced by Method A compared to Methods B and C and Control 1 methods. Specifically, the contents of all HGF, VEGF, progranulin, and exosomes were maximized in the cell supernatant produced by Method A.
本実施例の結果から、凍結解凍を行った細胞の培養上清には高濃度の生物活性物質が含まれることが示された。この効果は、解凍後少なくとも数代の継代を行った後の細胞を使用して培養上清を生産することが好ましいとする当該技術分野の技術常識に反する驚くべき結果である。 The results of this example showed that the culture supernatant of cells subjected to freezing and thawing contained a high concentration of biologically active substances. This effect is a surprising result contrary to the common general knowledge in the technical field that it is preferable to produce the culture supernatant using cells that have been passaged for at least several generations after thawing.
<実施例2:解凍後の培養条件>
(目的)
解凍後の細胞を培養する条件をさらに検討する。具体的には、解凍後の細胞を培養する培地の種類、及び継代の有無による、培養上清中の生物活性物質含有量に対する影響を検討する。
<Example 2: Culture conditions after thawing>
(the purpose)
The conditions for culturing cells after thawing are further examined. Specifically, the influence of the type of culture medium for culturing cells after thawing and the presence or absence of passage on the content of biologically active substances in the culture supernatant will be examined.
(方法)
凍結及び解凍を含む方法(方法D~F)、及び凍結及び解凍を含まない方法(対照2)の各方法により培養上清を生産した。各々の方法に含まれる各工程を以下で説明し、その概要を図5に示す。
(Method)
Culture supernatants were produced by methods that included freezing and thawing (Methods DF) and methods that did not include freezing and thawing (Control 2). Each step involved in each method is described below and outlined in FIG.
方法D:方法Dは、実施例1に記載の、(1)前培養、(2)細胞の凍結、(3)細胞の解凍、(4)無血清培地及び/又は血清含有培地での培養の(4-2)方法B、(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む。但し、(4-2)方法Bにおいて、血清含有培地及び無血清培地での培養時間はそれぞれ2日間とする(図5、「方法D」)。 Method D: Method D consists of (1) pre-culturing, (2) cell freezing, (3) cell thawing, and (4) culture in serum-free and/or serum-containing media as described in Example 1. (4-2) method B, (5) culture in basal medium, and (6) recovery of culture supernatant. However, in (4-2) Method B, culture time in serum-containing medium and serum-free medium is 2 days each (Fig. 5, "Method D").
方法E:方法Eは、実施例1に記載の、(1)前培養、(2)細胞の凍結、(3)細胞の解凍、(4)無血清培地及び/又は血清含有培地での培養の(4-1)方法A、(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む。但し、(4-1)方法Aにおいて、無血清培地での培養時間は4日間とした(図5、「方法E」)。 Method E: Method E is described in Example 1, (1) pre-culture, (2) cell freezing, (3) cell thawing, (4) culture in serum-free medium and/or serum-containing medium. (4-1) Method A, (5) culture in basal medium, and (6) recovery of culture supernatant. However, in (4-1) Method A, culture time in serum-free medium was 4 days (Fig. 5, "Method E").
方法F:方法Fは、方法Eにおいて無血清培地での培養に代えて血清含有培地での培養を行う方法である(図5、「方法F」)。血清含有培地は、FBSを添加したPRIME-XV MSC Expansion XSFMを使用し、培養時間は4日間とした。 Method F: Method F is a method of culturing in a serum-containing medium instead of culturing in a serum-free medium in Method E (Fig. 5, "Method F"). PRIME-XV MSC Expansion XSFM supplemented with FBS was used as the serum-containing medium, and culture time was 4 days.
凍結及び解凍を含まない方法(対照2):対照として行った方法は、実施例1に記載の(2)細胞の凍結及び(3)細胞の解凍を含まない。この方法(対照2)は、(1)前培養の後に無血清培地で培養を行い、次いで(5)基本培地での培養、及び(6)培養上清の回収の各工程を含む。但し、(1)前培養の培養期間は2日間とし、無血清培地での培養期間は2日間とした(図3、「対照2」)。 Freeze-and-thaw-free method (Control 2): The method performed as a control does not involve (2) freezing the cells and (3) thawing the cells as described in Example 1. This method (Control 2) includes the steps of (1) culturing in a serum-free medium after preculturing, then (5) culturing in a basal medium, and (6) collecting the culture supernatant. However, (1) the culture period for the preculture was 2 days, and the culture period in the serum-free medium was 2 days (Fig. 3, "Control 2").
(結果)
方法D~F、及び対照2の各方法により生産した培養上清において生物活性物質の含有量を測定した結果を図6に示す。
(result)
FIG. 6 shows the results of measuring the content of the biologically active substance in the culture supernatants produced by methods D to F and Control 2.
図6に示す結果から、方法Fと比較して、方法D及びEによって生産した細胞上清においてHGFの含有量が大幅に増加することが判明した。よって、解凍後の培養が無血清培地での培養を含むことにより、生物活性物質含量が顕著に増大する効果が示された。 The results shown in FIG. 6 revealed that the content of HGF was significantly increased in cell supernatants produced by methods D and E compared to method F. Therefore, the effect of significantly increasing the bioactive substance content was demonstrated by including culture in a serum-free medium after thawing.
また、図6に示す結果において、方法Dと方法Eの結果を比較すると、解凍後の培養を同じ期間行う場合には、無血清培地での培養の前に血清含有培地での培養を行う方が生物活性物質含量を増大し得ることも示された。 In the results shown in Fig. 6, comparing the results of Method D and Method E, it was found that culturing in a serum-containing medium was performed before culturing in a serum-free medium when culturing after thawing was performed for the same period of time. It has also been shown that can increase the bioactive substance content.
Claims (17)
凍結した細胞を解凍する、解凍工程、
前記細胞を第一の培地で培養する、第一培養工程、
前記第一培養工程後に得られる培養上清を除去する、除去工程、
前記除去工程後の前記細胞をさらに第二の培地で培養する、第二培養工程、及び
前記第二培養工程後に得られる培養上清を回収する、回収工程
を含み、
前記第一の培地が、基本培地又は無血清培地であり、かつ
前記第二の培地が、タンパク質を含まない、前記方法。 A method of producing a culture supernatant, comprising:
thawing the frozen cells, a thawing step;
a first culturing step of culturing the cells in a first medium;
a removing step of removing the culture supernatant obtained after the first culturing step;
a second culturing step of further culturing the cells after the removing step in a second medium; and a collecting step of collecting the culture supernatant obtained after the second culturing step,
The method as described above, wherein the first medium is a basal medium or a serum-free medium, and the second medium is protein-free.
前培養培地で細胞を培養する、前培養工程、
前記前培養工程後の前記前培養培地を凍結保存液と置換する、置換工程、及び
前記置換工程後の前記細胞を凍結する、凍結工程
を含み、
前記前培養培地が、血清又は血漿含有培地、無血清培地、又は基本培地である、請求項1~9のいずれか一項に記載の方法。 Before the thawing step,
a pre-culture step of culturing cells in a pre-culture medium;
A replacement step of replacing the pre-culture medium after the pre-culture step with a cryopreservation solution, and a freezing step of freezing the cells after the replacement step,
A method according to any one of claims 1 to 9, wherein the pre-culture medium is a serum- or plasma-containing medium, a serum-free medium, or a basal medium.
凍結した細胞を解凍する、解凍工程、
前記細胞を第一の培地で培養する、第一培養工程、
前記第一培養工程後に得られる培養上清を除去する、除去工程、
前記除去工程後の前記細胞をさらに第二の培地で培養する、第二培養工程、及び
前記第二培養工程後に得られるサイトカイン及び/又はエクソソームを含む培養上清を回収する、回収工程
を含み、
前記第一の培地が、基本培地又は無血清培地であり、かつ
前記第二の培地が、タンパク質を含まない、前記方法。 A method for increasing the content of cytokines and / or exosomes in the culture supernatant,
thawing the frozen cells, a thawing step;
a first culturing step of culturing the cells in a first medium;
a removing step of removing the culture supernatant obtained after the first culturing step;
Further culturing the cells after the removal step in a second medium, a second culturing step, and recovering the culture supernatant containing cytokines and / or exosomes obtained after the second culturing step, including a recovery step,
The method as described above, wherein the first medium is a basal medium or a serum-free medium, and the second medium is protein-free.
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